Sedimentation field flow frctionation is a versatile technique for the high resolution separation of a wide variety of particulates suspended in a fluid medium. The particulates include macromolecules in the 10.sup.5 to the 10.sup.13 molecular weight (0.001 to 1 .mu.m) range, colloids, particles, unicelles, organelles and the like. The technique is more explicitly described in U.S. Pat. No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Pat. No. 3,523,610, issued August 11, 1970 to Edward M. Purcell and Howard C. Berg.
Field flow fractionation is the result of the differential migration rate of components in a carrier or mobile phase in a manner similar to that of chromatography. However, in field flow fractionation there is no separate stationary phase as is in the case of chromatography. Sample retention is caused by the redistribution of sample components between the fast to the slow moving strata within the mobile phase. Thus, particulates elute more slowly than the solvent front. Typically a field flow fractionation channel consisting of two closely spaced parallel surfaces is used. A mobile phase is caused to flow continuously through the gap between the surfaces. Because of the narrowness of this gap or channel (typically 0.025 centimeters (cm)) the mobile phase flow is laminar with a characteristic parabolic velocity profile. The flow velocity is the highest at the middle of the channel and the lowest near the two channel surfaces.
An external force field of some type (the force fields include gravitational, thermal, electrical, fluid cross-flow and others as described variously by Giddings and Berg and Purcell), is applied transversely (perpendicular) to the channel surfaces or walls. This force field pushes the sample components in the direction of the slower moving liquid strata near the outer wall. The buildup of sample concentration near the wall, however, is resisted by the normal diffusion of the particulates in a direction opposite to the force field. This results in a dynamic layer of component particles, each component with an exponential--concentration profile. The extent of retention is determined by the particulate's time-average position within the concentration profile, which is a function of the balance between the applied field strength and the opposing tendency of particles to diffuse.
In sedimentation field flow fractionation, use is made of a centrifuge to establish the force field required for the separation. For this purpose a long, thin annular belt-like channel is made to rotate within a centrifuge. The resultant centrifugal force causes components of higher density than the mobile phase to settle toward the outer wall of the channel. For equal particle density, because of their higher diffusion rate, smaller particulates will accumulate into a thicker layer against the outer wall than will larger particles. On the average, therefore, larger particulates are forced closer to the outer wall.
If now the fluid medium, which may be termed a mobile phase or solvent is fed continuously in one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile within the channel and the placement of particulates in that profile, solvent flow causes smaller particulates to elute first, followed by a continuous elution of sample components in the order of ascending particulate mass.
As a general rule relatively long channels are required in many separations. Unfortunately, however, when Giddings et al. (J. C. Giddings, F. J. F. Yang, and M. N. Myers, Anal. Chem. 46, 1917 (1974)) tried such a long column using a coiled tubing, they failed apparently because the circular cross-section incurred secondary flow effects. Secondary flow is a component of flow that is normal to the column axis of a curved tube, and is induced by the increased centrifugal force experienced by fluid moving in the fast flow stream lines. Because of this effect there is a continuous recirculation of liquid within the tubing and if it is sufficiently strong, it recirculates the component peak causing a loss of retention and relatively large increases in band spreading. Giddings et al. propose that this secondary effect can be reduced by utilizing columns with a rectangular cross-section and having a large width to thickness ratio. Because of this early teaching, previous sedimentation field flow fractionations have been carried out using channels with a large width to thickness aspect ratio typically in a range of 50 to 200 to 1.
Channels having such large aspect width to thickness ratios increase the volume of fluid medium required to flow through the channel and decrease detection sensitivity since sample peaks can become diluted because of the large fluid volumes.